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International audience; Nosema ceranae is an emerging and invasive gut pathogen in Apis mellifera and is considered as a factor contributing to the decline of honeybee populations. Here, we used a combined LC-MS and NMR approach to reveal the metabolomics changes in the hemolymph of honeybees infected by this obligate intracellular parasite. For metabolic profiling, hemolymph samples were collected from both uninfected and N. ceranae-infected bees at two time points, 2 days and 10 days after the experimental infection of emergent bees. Hemolymph samples were individually analyzed by LC-MS, whereas each NMR spectrum was obtained from a pool of three hemolymphs. Multivariate statistical PLS-DA models clearly showed that the age of bees was the parameter with the strongest effect on the metabolite profiles. Interestingly, a total of 15 biomarkers were accurately identified and were assigned as candidate biomarkers representative of infection alone or combined effect of age and infection. These biomarkers included carbohydrates (α/β glucose, α/β fructose and hexosamine), amino acids (histidine and proline), dipeptides (Glu-Thr, Cys-Cys and γ-Glu-Leu/Ile), metabolites involved in lipid metabolism (choline, glycerophosphocholine and O-phosphorylethanolamine) and a polyamine compound (spermidine). Our study demonstrated that this untargeted metabolomics-based approach may be useful for a better understanding of pathophysiological mechanisms of the honeybee infection by N. ceranae.

International audience; Due to the widespread use of pesticides and their persistence in the environment, non-target organisms are chronically exposed tomixtures of toxic residues. Fungicides, herbicides and insecticides are all found at low doses in the diet of pollinators such as honey bees, but due to the lack of data on the toxicological effects of these mixtures, determining their risk is difficult to assess.We therefore developed a study combining the identification of common pollen-bound pesticide mixtures associated with poor colony development and tested their effects on bee behavior and physiology.Weexposed bees to the identified pesticidemixtures during the first days of their adult life, a crucial period for physiological development. Using optic bee counters we recorded the behavior of bees throughout their lives and identified two pesticidemixtures that delay the onset of foraging and slow-down foraging activity. Furthermore, one of thesemixtures hampers pollen foraging. As bee longevity is strongly influenced by the time spent foraging, bees exposed to these pesticide mixtures outlived control bees. Physiological analysis revealed that perturbations of the energeticmetabolism preceded the altered behavior. In conclusion,we found that early-life exposure to lowdoses of pesticidemixtures can have long-termeffects that translate into longer-lived but slower and less efficient bees. These surprising findings contrast with the commonly reported increase in bee mortality upon pesticide exposure, and demonstrate that exposure that may seem harmless (e.g., very low doses, pesticides not intended to kill insects) can have undesirable effects on non-target organisms.

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International audience; Modern agriculture often involves the use of pesticides to protect crops. These substances are harmful to target organisms (pests and pathogens). Nevertheless, they can also damage non-target animals, such as pollinators and entomophagous arthropods. It is obvious that the undesirable side effects of pesticides on the environment should be reduced to a minimum. Western honey bees (Apis mellifera) are very important organisms from an agricultural perspective and are vulnerable to pesticide-induced impacts. They contribute actively to the pollination of cultivated crops and wild vegetation, making food production possible. Of course, since Apis mellifera occupies the same ecological niche as many other species of pollinators, the loss of honey bees caused by environmental pollutants suggests that other insects may experience a similar outcome. Because pesticides can harm honey bees and other pollinators, it is important to register pesticides that are as selective as possible. In this manuscript, we describe a selection of methods used for studying pesticide toxicity/selectiveness towards Apis mellifera. These methods may be used in risk assessment schemes and in scientific research aimed to explain acute and chronic effects of any target compound on Apis mellifera.

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BACKGROUND Thymol offers an attractive alternative to synthetic chemicals to keep Varroa under control. However, thymol accumulates in bee products and is suspected of having adverse effects on colonies and especially on larvae. In this study, we investigated the effects of acute and chronic exposure to thymol on larvae reared in vitro with contaminated food and compared results to the theoretical larval exposure based on the amount of pollen and honey consumed by larvae during their development. RESULTS The laboratory assays reveal that, first, the 48 h-LD50 of thymol introduced into larval food is 0.044 mg larva−1. Second, the 6 day-LC50 is 700 mg kg−1 food. A significant decrease of larval survival and mass occurred from 500 mg thymol kg−1 food (P < 0.0001). Finally, vitellogenin expression, which reached a maximum at the fifth instar larvae, is delayed for individuals exposed to 50 mg thymol kg−1 food (P < 0.0006). That is 10 times higher than the theoretical level of exposure. CONCLUSION Based on the level of thymol residue found in honey and pollen, these results suggest that the contamination of food by thymol represents no notable risk for the early-developing larvae. © 2013 Society of Chemical Industry

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In France, a derogation to pesticide regulation allows spraying on blooms with insecticides or acaricides bearing the bee label (mention « Abeilles »), but only when no bees are foraging on crops. Nevertheless, no decision rule is available for farmers to assess bees’ absence on the crop. To fill this gap, the Ministry of Agriculture initiated a reflection about environmental and climatic factors influencing the presence of forager bees. This review takes an inventory of data available about foraging activity according to environment or weather. Decision rules based on such criteria appear inappropriate. Only nocturnal sprays of pesticides on crops significantly reduce the risk of bees’ exposure to chemicals. Fil: Decourtye, Axel. Institut National de la Recherche Agronomique; Francia Fil: Vidau, Cyril. Institut National de la Recherche Agronomique; Francia Fil: Rollin, Orianne. Institut National de la Recherche Agronomique; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte; Argentina Fil: Requier, Fabrice. Institut National de la Recherche Agronomique; Francia Fil: Rüger, Charlotte. Institut National de la Recherche Agronomique; Francia Fil: Allier, Fabrice. Institut National de la Recherche Agronomique; Francia Fil: Le Féon, Violette Alice Marie. Institut National de la Recherche Agronomique; Francia Fil: Kretzschmar, André. Institut National de la Recherche Agronomique; Francia Fil: Devillers, James. Institut National de la Recherche Agronomique; Francia Fil: Henry, Mickaël. Institut National de la Recherche Agronomique; Francia Fil: Odoux, Jean François. Institut National de la Recherche Agronomique; Francia

International audience; Microsporidia are emerging fungi-like intracellular parasites of economic, veterinary and medical importance. The strategy they use to invade their host is related to the rapid extrusion of a unique and highly specialized organelle, the polar tube, which allows the injection of the infectious spore content within a target cell. This original process seems to be dependent on initial interactions between parasite and host cell components. The extreme reduction and compaction of most microsporidian genomes resulted in the loss of many metabolic pathways, which makes these parasites highly dependent on their host. Recent significant advances have been made in the understanding of mammal and insect immune responses against microsporidian infections with the involvement of both adaptive and innate immunity.

Phenylpyrazoles are relatively new insecticides designed to manage problematic insect resistance and public health hazards encountered with older pesticide families. In vitro cytotoxicity induced by the phenylpyrazole insecticides, Ethiprol and Fipronil, and Fipronil metabolites, sulfone and sulfide, was studied in Caco-2 cells. This cellular model was chosen because it made possible to mimic the primary site of oral exposure to xenobiotics, the intestinal epithelium. Assessment of the barrier function of Caco-2 epithelium was assessed by TEER measurement and showed a major loss of barrier integrity after exposure to Fipronil and its metabolites, but not to Ethiprol. The disruption of the epithelial barrier was attributed to severe ATP depletion independent of cell viability, as revealed by LDH release. The origin of energetic metabolism failure was investigated and revealed a transient enhancement of tetrazolium salt reduction and an increase in lactate production by Caco-2 cells, suggesting an increase in glucose metabolism by pesticides. Cellular symptoms observed in these experiments lead us to hypothesize that phenylpyrazole insecticides interacted with mitochondria

International audience; Many invasive pathogens effectively bypass the insect defenses to ensure the completion of their life cycle. Among those, an invasive microsporidian species, Nosema ceranae, can cause nosemosis in honeybees. N. ceranae was first described in the Asian honeybee Apis cerana and is suspected to be involved in Western honeybee (Apis mellifera) declines worldwide. The midgut of honeybees is the first barrier against N. ceranae attacks. To bring proteomics data on honeybee/N. ceranae crosstalk and more precisely to decipher the worker honeybee midgut response after an oral inoculation of N. ceranae (10 days postinfection), we used 2D-DIGE (2-Dimensional Differential In-Gel Electrophoresis) combined with mass spectrometry. Forty-five protein spots produced by the infected worker honeybee group were shown to be differentially expressed when compared to the uninfected group; 14 were subsequently identified by mass spectrometry. N. ceranae mainly caused a modulation of proteins involved in three key host biological functions: (i) energy production, (ii) innate immunity (reactive oxygen stress) and (iii) protein regulation. The modulation of these host biological functions suggests that N. ceranae creates a zone of ‘‘metabolic habitat modification’’ in the honeybee midgut favoring its development by enhancing availability of nutrients and reducing the worker honeybee defense.

Données supplémentaires disponibles sur le site de la revue: http://www.journals.elsevier.com/infection-genetics-and-evolution/; International audience; Genetic diversity of a host species is a key factor to counter infection by parasites. Since two separation events and the beginning of beekeeping, the Western honeybee, Apis mellifera, has diverged in many phylogenetically-related taxa that share common traits but also show specific physiological, behavioural and morphological traits. In this study, we tested the hypothesis that A. mellifera taxa living in a same habitat should respond differently to parasites like Nosema ceranae, a microsporidia living in host's midgut. We used the Poulin and Combes' concept of virulence to compare the susceptibility of three A. mellifera taxa to N. ceranae infection. Three criteria were measured 10 days post-infection (dpi): the host mortality, the host sugar consumption and the development success of the parasite (i.e. number of spores produced). Interestingly, we showed that the observed variation in susceptibility to infection by N. ceranae is not linked to honeybee taxa but results from the variability between colonies, and that those differences are probably linked to genetic variations. The use of these three criteria allows us to conclude that the differences in susceptibility are mediated by a genetic variability in honeybee workers from resistance to tolerance. Finally, we discuss the consequences of our findings for beekeeping management.

Thymol offers an attractive alternative to synthetic chemicals to keep Varroa under control. However, thymol accumulates in bee products and is suspected of having adverse effects on colonies and especially on larvae. In this study, we investigated the effects of acute and chronic exposure to thymol on larvae reared in vitro with contaminated food and compared results to the theoretical larval exposure based on the amount of pollen and honey consumed by larvae during their development.The laboratory assays reveal that, first, the 48 h-LD50 of thymol introduced into larval food is 0.044 mg larva(-1) . Second, the 6 day-LC50 is 700 mg kg(-1) food. A significant decrease of larval survival and mass occurred from 500 mg thymol kg(-1) food (P0.0001). Finally, vitellogenin expression, which reached a maximum at the fifth instar larvae, is delayed for individuals exposed to 50 mg thymol kg(-1) food (P0.0006). That is 10 times higher than the theoretical level of exposure.Based on the level of thymol residue found in honey and pollen, these results suggest that the contamination of food by thymol represents no notable risk for the early-developing larvae.

International audience; Pesticides and pathogens are two categories of environmental stressors that may contribute to the decline of honey bee populations (vanEngelsdorp and Meixner, 2010). However, if their separate impacts on the honey bee are relatively well studied, knowledge on their interactions are somewhat lacking. Pioneer studies on toxico-pathological interactions have been conducted on the association of Nosema and chronic bee paralysis virus (CBPV) with organophosphate, organochlorine and pyrethroid insecticides (Ladas, 1972; Bendahou et al., 1997). These studies focused on the acute exposure to insecticides regardless of their chronic toxicity. However, the introduction of systemic insecticides, such as phenylpyrazoles and neonicotinoids in the mid 1990's renders more relevant the studies on chronic exposures to pesticides by oral route. Since, Suchail et al. (2001) have reported a discrepancy between acute and chronic toxicity of the neonicotinoids imidacloprid and its metabolites showing a high toxicity at very low doses.

International audience; In ecosystems, a variety of biological, chemical and physical stressors may act in combination to induce illness in populations of living organisms. While recent surveys reported that parasite-insecticide interactions can synergistically and negatively affect honeybee survival, the importance of sequence in exposure to stressors has hardly received any attention. In this work, Western honeybees (Apis mellifera) were sequentially or simultaneously infected by the microsporidian parasite Nosema ceranae and chronically exposed to a sublethal dose of the insecticide fipronil, respectively chosen as biological and chemical stressors. Interestingly, every combination tested led to a synergistic effect on honeybee survival, with the most significant impacts when stressors were applied at the emergence of honeybees. Our study presents significant outcomes on beekeeping management but also points out the potential risks incurred by any living organism frequently exposed to both pathogens and insecticides in their habitat.

Fipronil is a phenylpyrazole insecticide known to elicit neurotoxicity via an interaction with ionotropic receptors, namely GABA and glutamate receptors. Recently, we showed that fipronil and other phenylpyrazole compounds trigger cell death in Caco-2 cells. In this study, we investigated the mode of action and the type of cell death induced by fipronil in SH-SY5Y human neuroblastoma cells. Flow cytometric and western blot analyses demonstrated that fipronil induces cellular events belonging to the apoptosis process, such as mitochondrial potential collapse, cytochrome c release, caspase-3 activation, nuclear condensation and phosphatidylserine externalization. In addition, fipronil induces a rapid ATP depletion with concomitant activation of anaerobic glycolysis. This cellular response is characteristic of mitochondrial injury associated with a defect of the respiration process. Therefore, we also investigated the effect of fipronil on the oxygen consumption in isolated mitochondria. Interestingly, we show for the first time that fipronil is a strong uncoupler of oxidative phosphorylation at relative low concentrations. Thus in this study, we report a new mode of action by which the insecticide fipronil could triggers apoptosis.

International audience; BACKGROUND: The honeybee, Apis mellifera, is undergoing a worldwide decline whose origin is still in debate. Studies performed for twenty years suggest that this decline may involve both infectious diseases and exposure to pesticides. Joint action of pathogens and chemicals are known to threaten several organisms but the combined effects of these stressors were poorly investigated in honeybees. Our study was designed to explore the effect of Nosema ceranae infection on honeybee sensitivity to sublethal doses of the insecticides fipronil and thiacloprid. METHODOLOGY/FINDING: Five days after their emergence, honeybees were divided in 6 experimental groups: (i) uninfected controls, (ii) infected with N. ceranae, (iii) uninfected and exposed to fipronil, (iv) uninfected and exposed to thiacloprid, (v) infected with N. ceranae and exposed 10 days post-infection (p.i.) to fipronil, and (vi) infected with N. ceranae and exposed 10 days p.i. to thiacloprid. Honeybee mortality and insecticide consumption were analyzed daily and the intestinal spore content was evaluated 20 days after infection. A significant increase in honeybee mortality was observed when N. ceranae-infected honeybees were exposed to sublethal doses of insecticides. Surprisingly, exposures to fipronil and thiacloprid had opposite effects on microsporidian spore production. Analysis of the honeybee detoxification system 10 days p.i. showed that N. ceranae infection induced an increase in glutathione-S-transferase activity in midgut and fat body but not in 7-ethoxycoumarin-O-deethylase activity. CONCLUSIONS/SIGNIFICANCE: After exposure to sublethal doses of fipronil or thiacloprid a higher mortality was observed in N. ceranae-infected honeybees than in uninfected ones. The synergistic effect of N. ceranae and insecticide on honeybee mortality, however, did not appear strongly linked to a decrease of the insect detoxification system. These data support the hypothesis that the combination of the increasing prevalence of N. ceranae with high pesticide content in beehives may contribute to colony depopulation.

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International audience; Chaque année du début du prin - temps jusqu'aux premiers jours de l'automne, des milliards d'abeilles sortent de leurs ruches et partent visiter les fleurs des paysages environnants. Elles y trouvent nectars et pollens, éléments essentiels à la base de leur alimentation. C'est par cette intense activité de butinage que l'abeille domestique participe activement à la pollinisation des plantes sauvages et cultivées. Son influence directe sur la qualité et la quantité des récoltes ainsi que sur le maintien de la biodiversité florale est aujourd'hui largement validée et souligne le rôle prépondérant des abeilles (sauvages et domestiques) dans le fonctionnement des écosystèmes. En France, les abeilles domestiques, Apis mellifera (Hymenoptera: Apidae), sont l'outil de travail d'environ 70 000 apiculteurs professionnels et amateurs qui tirent bénéfice des labeurs de cet hyménoptère (environ 20 000 tonnes de miel récoltées en France en 2009).